GAS1, GAS2, GAS3 and GAS4 : four developmentally regulated genes with specialized roles at different stages of the yeast life cycle

The formation of Saccharomyces cerevisiae cell wall requires the coordinated activity of enzymes involved in the biosynthesis and modification of its components, such as glucans. The β-(1,3)-glucan synthase complexes, that have Fks proteins as putative catalytic subunits, use UDP-glucose as a substrate and catalyse the synthesis and vectorial extrusion of glucan chains into the outer space. Then, β-(1,3)-glucan chains are branched, elongated and remodelled in order to create a robust texture capable of counteracting the high internal pressure and determining cell morphology. β-(1,3)-glucan is the main component of the vegetative cell wall and one of the most abundant polymers of the spore wall. Several enzymes belonging to the family GH72 of glycosyl hydrolases have been identified in fungi. These enzymes are responsible of the lateral elongation of β-(1,3)-glucan, thus contributing to the assembly and organization of the glucan layer. The multigene GAS family of S. cerevisiae is composed of five members, GAS1-5, involved in cell wall maintenance. They share significant similarity with Aspergillus fumigatus GEL1 and GEL2, and with Candida albicans PHR1 and PHR2. Similar to the most extensively characterized member, Gas1p, the remaining Gas proteins are β-(1,3)-glucanosyltransferases involved in cell wall assembly and maintenance. Based on their expression patterns, they appear to play partially overlapping roles throughout the yeast life cycle: GAS1 and GAS5 are expressed during vegetative growth, whereas GAS2 and GAS4 are expressed exclusively during sporulation and required for normal spore wall formation, finally GAS3 is a weakly expressed gene. Thus these enzymes could satisfy the cellular needs to remodel β-(1,3)-glucan in different physiological conditions and in different conformations along the yeast life cell cycle. Moreover, considering its role in yeast cell biology, the GAS enzyme family represents a very promising molecular target for new antifungal drugs. During my PhD thesis I focused my interest on the functional characterization of GAS1, 2, 3 and 4 in various stages of the yeast life cycle: vegetative growth, meiosis, sporulation and spore germination. This study is aimed to understand the biological significance of the developmentally regulated requirement of the specific members of the GAS redundant family in the morphological stages of yeast life cycle. GAS2 and GAS4 genes are specifically induced during sporulation and encode for glycoproteins. The effects of the loss of Gas2 and Gas4 proteins on spore wall morphogenesis are dramatic. Synthesis of all the layers of the spore cell wall occurs, but the accumulation and organization of wall material is abnormal. The lack of the elongase activity of Gas2 and Gas4 proteins in the double mutant might cause the formation of shorter or less branched β(1,3)-glucan chains in the inner layer of the spore wall. Thus, the connection of the outermost layers to a less compact glucan network could make the spore wall more fragile and easily stripped under harsh conditions. These defects cause an increase in spore permeability to exogenous substances, a decrease in refractivity, and a marked decrease in spore viability. The possible execution point for GAS2 and GAS4 could be between the synthesis and organization of β(1,3)-glucan and, more specifically, in the elongation of the β(1,3)-glucan chains. Consistently with their role, during sporulation Gas2 and Gas4 proteins localize at the newly assembling prospore membrane during the meiotic divisions and in mature ascospore the proteins decorate the spore periphery. A slight difference in the protein patterns of fluorescence on the spore suggests that Gas2p and Gas4p final localization could be respectively the spore wall and the prospore membrane. In this work, an extensive study of the localization of the Gas1 protein during the yeast life cycle was performed, taking advantage of a GFP-tagged version of the protein. During vegetative growth Gas1p has a dual localization: in the plasma membrane and at the site of bud emergence, particularly in the neck, in the chitin ring that surrounds the neck region and in the bud scars where Gas1p remains after cytokinesis. At the neck region Gas1p appears to absolve important functions in yeast as a part of the mechanisms that ensure the resistance of the neck region and the morphogenesis of the septum. The size and morphology of the neck region is severely affected both in the gas1Δ and gas1Δ chs3Δ mutant, suggesting an involvement of the protein in the maintenance of the integrity of the mother-bud neck region. The presence of Gas1p in the chitin ring could be part of the mechanism necessary to prevent new incorporation of glucan chains into the neck region or alternatively the protein could be required for a particular type of remodelling necessary for the septum region in preparation to cell division. Additionally, Gas1p could act as landmark protein for the choice of the site of bud emergencee. As to Gas1p localization at the plasma membrane, our study supports the validity of Gas1p-GFP as a marker to follow the dynamics of lipid raft. At the induction of sporulation, GAS1 mRNA levels steadily decrease and by 10h it is completely declined. Surprisingly, Gas1p levels are roughly constant during the entire sporulation processs and the protein is very stable, being detectable also at 43h after the induction of sporulation. During spore development, a translocation event occurs through which at the completion of meiosis II, Gas1p, synthesized during vegetative growth, is removed from the plasma membrane and internalized. Later, Gas1p is detected associated to the nascent prospore membrane surrounding the nuclear lobes and finally in mature spores it localizes at the spore periphery. This translocation event suggests that Gas1p delivery to the spore surface is not part of the developmentally reprogramming of the secretory pathway from the trans Golgi to the prospore membrane, whereas it involves at least in part the endocytic pathway. We demonstrated that END3-mediated endocytosis is one of the mechanisms required for the removal of the Gas1p from the plasma membrane and its efficient re-localization at the prospore membrane. Moreover in a sps1Δ mutant, Gas1p remains localized at the plasma membrane and fails to reach the spore surface. Sps1p is a member of the Ste20 protein kinase family and regulates the trafficking to the prospore membrane of enzymes involved in spore wall synthesis, such as the glucan synthase Fks2p and chitin synthase Chs3p. Thus Sps1p could regulate the traffic of Gas1p most likely in an indirect way by interacting and modifying the components of the intracellular trafficking machinery. Gas1p translocation during sporulation To test a possible involvement of Gas1p in spore wall formation, in this study we tried to characterize the sporulation phenotype of a gas1Δ mutant. Unfortunately our analysis was complicated by the mutant reduced cell viability when grown in presence of a poor carbon source such as acetate. gas1Δ sporulation defect could rely in a unsatisfied energetic request as the cell wall perturbations, typical of a gas1Δ mutant, enhance carbon and energy mobilization to efficiently combat cell wall weakening and the metabolism of acetate as the sole carbon source could be not sufficient to satisfy this energetic request. Moreover the addition of sorbitol to the sporulation medium only partially rescues gas1Δ defective phenotype during spore development. Even though sorbitol can mitigate the gas1Δ cell wall damages, it has no buffering effect on the gas1Δ energetic request, thus the mutant cells remained substantially unable to sporulate. Consequently, gas1Δ sporulation defective phenotype appears to be reminiscent of the mutant defects during vegetative growth, even worsened in a poor carbon source. Even though we cannot exclude a role for Gas1p during spore morphogenesis, it is our consumption that the protein translocation to the spore represents a “storage”mechanisms to ensure the presence of the Gas1p during spore germination. At 3h after the shift to a rich medium, Gas1p exhibits a highly polarized distribution, decorating exclusively half of the germinating spore in its growing pole. The protein localization is consistent with its role in glucan layer remodelling of the cell wall at the growing portion of the germinating cell. Besides gas1Δ germinating spore inability to support the elongation during the polarized growth of the cell suggests that Gas1p is required for a very early step in germination. Besides the protein is involved in a post-germination stage to support the polarized growth of the newly emerging bud. Finally, in this study we reported the preliminary results about the functional characterization of GAS3. The gene is expressed at a very low level during the vegetative growth in glucose and acetate. Consistently with the GAS3 expression pattern, Gas3p appears as a highly polydispersed glycoprotein of high molecular weight that is present in vegetative growing cells and along the sporulation process. EndoH treatment reduces the size and the aspect of the protein to a sharp band, suggesting that Gas3p is a heavily N-glycosylated protein. The experiments indicated that neither the overexpression nor the deletion of the GAS3 gene, alone or in combination with GAS2 and GAS4, lead to relevant differences in sporulation with respect toh the wild type or with the defective phenotype of the gas2 gas4 null mutant strain . The construction of a tagged version of the Gas3 protein to determine its localization will be a useful tool to understand the function ofl Gas3p during yeast life cycle.